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β-1,3-Oligoglucan:orthophosphate glucosyltransferases from Euglena gracilis
BIOCHIMICA ET BIOPHYSICAACTA
431
BBA 65655 fl-I,3-OLIGOGLUCAN : O R T H O P H O S P H A T E GLUCOSYLTRANSFERASES FROM EUGLENA GRACILIS II. COMPARATIVE S T U D I E S B E T W E E N L A M I N A R I B I O S E - AND fl-I,3-OLIGOGLUCAN P H O S P H O R Y L A S E
LUIS R. MARECHAL Instituto de Investigaciones Bioqulmicas "Fundaci6n Campomar", Obligado 2490, Buenos Aires (28) (A rgentina) (Received May i2th, 1967)
SUMMARY
I. Further studies have been carried out on the specificity of laminaribiose- and fl-I,3-oligoglucan phosphorylase. Both enzymes, present in extracts of Euglena gracilis, could be separated either by calcium phosphate gel or by DEAE-cellulose column chromatography. 2. From the data presented it can be concluded that the two enzymes catalyze the same reaction but with different quantitative specificity. The fl-I,3-oligoglucan phosphorylase was found to phosphorolyze laminaritriose and higher homologues at a greater rate than laminaribiose while the opposite behavior was observed with laminaribiose phosphorylase. 3- Another, and probably more striking, difference between the two enzymes was the fact that aged preparations of fl-i,3-oligoglucan phosphorylase showed an absolute requirement of sulihydryl donors while laminaribiose phosphorylase was only slightly activated. 4. The possibility that the qualitative similarity might be due to a crosscontamination of the enzymatic fractions was discarded by competition experiments between substrates and b y the elution pattern of the DEAE-cellulose chromatography.
INTRODUCTION
Two separable enzyme activities on fl-I,3-oligoglucans are present in extracts of Euglena gracilis strain z: laminaribiose phosphorylase (fl-I,3-oligoglucan:orthophosphate glucosyltransferase I) and fl-I,3-oligoglucan phosphorylase (/5-I,3-oligoglucan:orthophosphate glucosyltransferase II). In the preceding communication 1, some properties of the latter enzyme were described, as well as its separation from laminaribiose phosphorylase. Biochim. Biophys. Acta, 146 (1967) 431-4¢2
432
L . R . MARECHAL
Since both phosphorylases catalyze the same reaction, (fl-I,3-Glc)n + Pi ~ (fl-I,3-Glc)n-i + a-D-Glc-I-P
a comparative study between both enzymes has now been carried out in order to gain further insight into their similarities and differences. In addition, a method for the separation of the two enzymes by DEAE-cellulose column chromatography is reported. EXPERIMENTAL PROCEDURE
Most of the materials and methods used were the same as previously described 1 except for those specifically mentioned.
Enzyme assay Unless otherwise specified they were as follows: Laminaribiose phosphorylase. Both Procedures* A and B, detailed in the previous work, have been used except that 5 #moles of glucose were used instead of laminaribiose in Procedure A, and 0.5/,mole of the latter sugar was substituted for laminaritriose in Procedure B. Controls were carried out similarly but omitting the sugars. One unit of enzyme is defined as that amount catalyzing the formation of I/~mole of Pi per min under the conditions described in Procedure A**. fl-z,3-oligoglucan phosphorylase. Either Procedure A or B, described previously i, was used to measure its activity. RESULTS
Enzyme preparation from calcium phosphate gel Cells of Euglena gracilis strain z were grown, harvested and processed as described in the preceding paper i. The first fraction eluted from the calcium phosphate gel with 5 mM sodium pyrophosphate (Fraction I, containing o.oi to 0.04 units) was used as laminaribiose phosphorylase and the first fraction eluted with IO mM sodium pyrophosphate (Fraction IV, containing o.oi to 0.04 units) was employed as fl-I,3oligoglucan phosphorylase.
Enzyme preparation from DEAE-cellulose column chromatography Cells of the same organism grown in darkness were harvested and processed as described previously 2 up to the 33-66% ammonium sulfate step. The latter fraction (13 ml, 50 mg protein per ml) was dialyzed overnight against io mM TrisHCI-I mM EDTA, pH 7.2, and then applied to a column of DEAE-cellulose (16 cm ~ × 32 cm) equilibrated with the same buffer. All operations were carried out at o-4 °. After the protein was passed through the column, the latter was washed with IOO ml of the same buffer, and then with ioo ml of o.i M NaC1 in IO mM Tris-HCl-i mM * The p r e i n c u b a t i o n of laminaribiose p h o s p h o r y l a s e w i t h m e r c a p t o e t h a n o l was n o t necess a r y for activity. However, it was carried out as usual in order to standardize the assay of b o t h phosphorylases. ** This definition of laminaribiose p h o s p h o r y l a s e u n i t is different from t h a t given previously 2.
Biochim. Biophys. Acta, 146 (1967) 431-442
/~-I,3-GLUCAN PHOSPHORYLASES FROM E. gracilis. II
f
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Fig. i. Chromatography on DEAE-cellulose. 13 ml of the "33-66% ammonium sulfate step" fraction2 was applied to a column (16 cmt × 32 cm) equilibrated with o.oi M Tris-HCl-o.ooI M EDTA, pH 7.2. A linear gradient of NaCI between o.I and 0.35 M in the equilibrium buffer was applied to the column (50o ml of each concentration) with a flow rate of 1-1. 5 ml/min. Fractions of approx. 5 ml were collected. The enzyme activity was measured as described in Enzyme assay, Procedure A. Acceptor: (V--IF) glucose; (Ill--B) laminaribiose. Fig. 2. DEAE-cellulose column chromatography of Peak II. 12 ml of the protein extract from Peak II (see text) were passed through a o.75 cm~ × 32 cm column of DEAE-cellulose equilibrated with o.oi M Tris-HCl-o.ooi M EDTA, pH 7.2. A linear gradient of NaC1 between o.i and 0.35 M in the equilibrium buffer (3° ml of each concentration) with a flow rate of 0.3 ml/min. Fractions of approx. 1.5 ml were collected. Symbols as in Fig. I. E D T A , p H 7.2. The protein was eluted with a linear gradient of NaC1 (from o.I M to 0.35 M c o n c e n t r a t i o n in the equilibrium buffer, 500 ml of each saline concentration). The fractions c o n t a i n i n g the peaks of a c t i v i t y were pooled (Fig. I) a n d 2.5 vol, of n e u t r a l s a t u r a t e d a m m o n i u m sulfate solution were added. The suspension was allowed to s t a n d overnight before centrifugation at i o ooo × g for 20 mill. The precipitate o b t a i n e d was dissolved in a small volume of i o mM T r i s - H C l - i mM E D T A buffer, p H 7.2, a n d dialyzed overnight against the same solution. Fig. I shows t h a t two similar a c t i v i t y peaks were o b t a i n e d using glucose as s u b s t r a t e ; one of t h e m (Peak I, laminaribiose phosphorylase) was eluted between o.18 a n d 0.23 M NaC1 a n d the other (Peak II, /5-I,3-oligoglucan phosphorylase) between 0.25 a n d 0.32 M NaC1. W h e n laminaribiose was used as acceptor, two a c t i v i t y peaks were also obtained, b u t the a c t i v i t y of Peak I was very small compared with Peak II. I t can also be observed t h a t the profile of activities o b t a i n e d with the two acceptors were coincident. I n order to discard the possibility t h a t the presence of two a c t i v i t y peaks m i g h t be due to some artifact in the column chromatography, the protein fraction originated from Peak I I was r e c h r o m a t o g r a p h e d in a 0.75 cm ~ × 32 cm DEAE-cellulose c o l u m n prepared as already m e n t i o n e d . Fig. 2 shows t h a t only one peak was o b t a i n e d which emerged at a p p r o x i m a t e l y the same saline c o n c e n t r a t i o n as previously. I t m u s t be m e n t i o n e d t h a t when a n a l y t i c a l DEAE-cellulose columns (0.75 cm 2 × 32 cm) were used, the laminaribiose phosphorylase was eluted between o.14 a n d o.19 M a n d the fi-I,3-oligoglucan phosphorylase emerged between 0.22 a n d 0.26 M NaC1. After the r e c h r o m a t o g r a p h y of Peak II, the active fractions were pooled, c o n c e n t r a t e d a n d dialyzed as m e n t i o n e d above. This p r e p a r a t i o n a n d t h a t corres p o n d i n g to Peak I were used as fi-I,3-oligoglucan phosphorylase a n d laminaribiose phosphorylase, respectively, to carry out some of the assays m e n t i o n e d below.
Comparative studies using enzymes separated with calcium phosphate gel Effect of mercaptoethanol concentration on the reaction rate of both enzymes. The different behaviors of the phosphorylases towards increasing concentrations of
Biochim. Biophys. Acta, 146 (1967) 431-442
434
L.R. MARECHAI
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Fig. 3. Influence of mercaptoethanol concentration on the activity of fl-I,3-oligoglucan and laminaribiose phosphorylase. (A) Laminaribiose (0. 5/,mole) as acceptor. The standard assay for each enzyme was as described in Procedure A, except that the -SH donor concentration was varied as shown. For the assay of fl-i,3-oligoglucan phosphorylase, Fraction IV was used and for that corresponding to laminaribiose phosphorylase, Fraction I was employed. (B) Glucose (5/*moles) as acceptor. The assays were similar to (A) except that another enzyme batch was used, and that the third fraction eluted with 5 mM sodium pyrophosphate (Fraction III) was employed for the assay of /~-i,3-oligoglucan phosphorylase. Symbols as in Fig. I. Solid line: fl-I,3-oligoglucan phosphorylase; broken line: laminaribiose phosphorylase. m e r c a p t o e t h a n o l using laminaribiose as acceptor is shown in Fig. 3A. I t can be seen t h a t t h e reaction rate for fl-I,3-oligoglucan phosphorylase was increased ab o u t Io-fold at o p t i m a l c o n c e n t r a t i o n o f - S H donor, while t h a t for laminaribiose phosphorylase r e m a i n e d unchanged. Similar effects were o b t a i n e d when glucose was used as acceptor (Fig. 3B) b u t only a 2-fold increase in the reaction rate for fl-I,3-oligoglucan phosphorylase was observed. This decrease in the a c t i v a t i o n m i g h t be due to a certain c o n t a m i n a t i o n of the l a t t e r e n z y m e p r e p a r a t i o n with laminaribiose phosphorylase, thus m a s k i n g the ex act a m o u n t of P1 released in absence of m e r c a p t o e t h a n o l (see below). Phosphorolysis of fl-z,3-oligoglucans. B o t h enzymes catalyze t h e phosphorolysis of several/~-I,3-oligoglucans, b u t the initial reaction rates were different. As shown in Table I, laminaribiose phosphorylase act ed on laminaribiose to form a - G l c - I - P , 3 - 4 times faster t h a n on higher oligosaccharides. Th e inverse was true for fl-I,3-oligoglucan phosphorylase. I t m u s t be p o i n t e d out t h a t l a m i n a r i h e p t a o s e was also phosphorolyzed b y laminaribiose phosphorylase. Thus, in an e x p e r i m e n t similar to t h a t described in TABLE I PHOSPHOROLYSIS OF • - I , 3 - O L I G O G L U C A N S
The test conditions were as described in Enzyme assay, Procedure B. The oligosaccharides indicated below (0. 5 #,mole of each) were added and the amount of Gle-I-P formed was measured with the phosphoglucomutase-Glc-6-P dehydrogenase-NADP system.
Substrate
Laminaribiose phosphorylase (mtzmoles Glc-I-P formed per IO rain)
fl-z,3-oligoglucan phosphorylase (mt~moles Glc-z-P formed per io rain)
Laminaripentaose Laminaxitetraose Laminaritriose Laminaribiose
34 44 46 17o
8o
80 80 2O
Biochim. Biophys. Acta, 146 (1967) 431-442
fl-I,3-GLUCAN PHOSPHORYLASESFROM E. gracilis. II
435
Table I and taking the amount of Glc-I-P formed with laminaribiose as IOO%, approx. 3% of the sugar phosphate came from I mM laminariheptaose. Under the same conditions, ~-I,3-oligoglucan phosphorylase formed 13o% of Glc-I-P.
Phosphorolysis of ~-glucosyl derivatives of hydroquinone and saligenin by laminaribiose phosphorylase. It was previously reported 2 that two or more additional products were formed when laminaribiose phosphorylase was incubated with Glc-I-P and several acceptors, as judged by paper chromatography. However, in the case of arbutin, salicin and ~-phenylglucoside only one additional spot was detected. It was suggested that another enzyme could be responsible for the different behavior of these acceptors. A further possibility could be that the higher homologues were not detected due to the scarcity of the material enzymatically produced. A new approach was then attempted taking advantage of the reversibility of the reaction and the higher sensibility of the method for Glc-I-P determination as compared to that for Pi. As shown in Table II, laminaritriosyl-hydroquinone, laminaritetraosyl-saligenin and laminaritriosyl-saligenin were phosphorolyzed by laminaribiose phosphorylase, but the reaction rate was about 9o% lower than for the phenolic monosaccharides. For comparison, the values obtained with laminaribiose and laminaritetraose are also given. Although the results are not conclusive, the possibility remains that laminaribiose phosphorylase is actually involved in the reaction. TABLE
II
P H O S P H O R O L Y S I S OF V A R I O U S S U B S T R A T E S W I T H L A M I N A R I B I O S E P H O S P H O R Y L A S E
The enzyme was assayed as described under Procedure B, except for the glucosyl donors that replace laminaribiose as detailed below. The Gle-I-P formed during the incubation was determined as described in Table I and the amount corresponding to laminaribiose was taken as lOO% of relative activity.
Glucosyl donor
Concentration (raM)
Relative activity
Laminaribiose Laminaribiosyl-hydroquinone Laminaribiosyl-saligenin Laminaritetraose Laminaritriosyl-hydroquinone Laminaritriosyl-saligenin Laminaritetraosyl-sa!igenin
io 8 8 13 8 8 7
IOO 13o
135 i8 IO
5 6
Glucose and several ~-~,3-oligoglucans as acceptors for both enzymes. Since both enzymes could phosphorolyze the same/5-I,3-oligo sugars, although at different rates, it was considered of interest to assay these compounds as acceptors. Fig. 4 shows that laminaribiose phosphorylase and/5-I,3-oligoglucan phosphorylase can actually also catalyze at different rates similar reactions in the direction of synthesis. Comparative studies using enzymes separated with DEAE-cellulose column chromatography Specificity. fl-I,3-oligoglucan phosphorylase originated from the rechromatography on DEAE-cellulose, was assayed with most of the acceptors previously used 1 and similar values for the liberation of Pi were obtained.
Biothim. Biophys. Acta, 146 (1967) 431-442
436
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Fig. 4. Glucose and several fl-i,3-oligoglucans as acceptors for laminaribiose and fl-i,3-oligoglucan phosphorylases. The enzymatic assays were as described u n d e r Procedure A for the acceptors indicated. The concentration of the sugars was 5 mM, except for laminariheptaose which was 2. 4 mM. After 15 min incubation, the Pl liberation was determined. Symbols and lines as in Fig. 3.
F r o m t h e results of Figs. i a n d 2, it a p p e a r s t h a t b o t h enzymes can use glucose a n d laminaribiose as aceeptor. H o w e v e r to exclude the possibility of a m u t u a l c o n t a m i n a t i o n , c o m p e t i t i o n e x p e r i m e n t s between acceptors were p e r f o r m e d as a new a p p r o a c h to solve this problem. The results are i l l u s t r a t e d in Figs. 5 a n d 6. The t h e o r e t i c a l curves were o b t a i n e d using the following equations according to D i x o n AND WEBB 3 for the d e t e r m i n a t i o n of t h e t o t a l v e l o c i t y (vt) when a single enzyme acts on two s u b s t r a t e s a a n d b Va-
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Laminoribiose (raM)
Fig. 5 - L a m i n a r i b i o s e phosphorylase. Competition between laminaribiose and laminaritriose. (A) The incubation m i x t u r e s and enzyme activity were carried out as indicated in E n z y m e assay, Procedure B, b u t containing ill addition laminaritriose ( O - - O ) as indicated. Laminaribiose phosphorylase originated from Peak I (Fig. I) (0.o07 units) was used to carry out these experiments. Under the same conditions, the Kra for laminaribiose and laminaritriose were I mM and 2.1 raM, and the Vmax. 4.6 and 1.2 mktmoles/min, respectively. (B) The conditions were similar to (A) b u t m a i n t a i n i n g the a m o u n t of laminaritriose (0. 5/*mole) c o n s t a n t and v a r y i n g t h a t of laminaribiose as indicated. Symbols as in Fig. I. Solid line represents the theoretical curve (see text). Fig. 6. fl-i,3-oligoglucan phosphorylase. Competition between glucose and laminaribiose. (A} The incubation m i x t u r e s and enzyme were carried o u t as indicated in E n z y m e assay, Procedure A, b u t containing glucose as indicated, fl-I,3-oligoglucan p h o s p h o r y l a s e originated from the rec h r o m a t o g r a p h y of Peak I I (0.04 unit) was used to carry out these experiments. U n d e r the same conditions, the K m for glucose and laminaribiose were 45 and 4 raM, and the Vmax. 41 and 47 m#moles/min, respectively. (B) Tile conditions were similar to (A) b u t w i t h 4 ° mM glucose and v a r y i n g the a m o u n t of laminaribiose as indicated. Solid line represents the theoretical curve. Symbols as in Fig. i. 13iochirn. Biophys. Acta, 146 (1967) 431-442
f l - I , 3 - G L U C A N PHOSPHORYLASES FROM E.
gracilis,
437
ii
b are the concentration of substrates, Va and Vb, their m a x i m u m Ka and K~, their Michaelis constants. It can be seen that the experiobtained with both phosphorylases fall on the calculated curves, thus presence of only one active site in each enzymatic preparation. Reaction products with glucose and laminaribiose as acceptors. It seemed interesting to compare the pattern of the reaction products obtained with both phosphorylases in view of their similar enzymic activities. For this purpose, laminaribioseand fl-I,3-oligoglucan phosphorylases were incubated separately with [14qGlc-r-P and glucose or laminaribiose as acceptor. The result of a radiochromatogram scanning after 15 and 45 rain incubation is depicted in Figs. 7 and 8. where a and velocities and mental points indicating the
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Fig. 7- R a d i o a c t i v e r e a c t i o n p r o d u c t s f r o m b o t h p h o s p h o r y l a s e s u s i n g glucose as acceptor. (A) L a m i n a r i b i o s e p h o s p h o r y l a s e o r i g i n a t e d f r o m P e a k I (Fig. I) w a s p r e i n c u b a t e d as described in t e x t a n d a f t e r w a r d s i n c u b a t e d w i t h i / ~ m o l e of glucose a n d 0.6/~mole of [14C]Glc-I-P (36 ooo c o u n t s / m i n ) for 15 rain. (B) fl-i,3-oligoglucan p h o s p h o r y l a s e o r i g i n a t e d f r o m t h e r e c h r o m a t o g r a p h y of P e a k II (Fig. 2) w a s p r e i n c u b a t e d as described in t e x t a n d a f t e r w a r d s i n c u b a t e d as detailed in (A) d u r i n g 15 min. (C) Similar to (A) b u t t h e i n c u b a t i o n t i m e w a s increased to 45 min. (D) Similar to (B) b u t t h e i n c u b a t i o n t i m e w a s increased to 45 rain. T h e controls were carried o u t as a b o v e b u t o m i t t i n g t h e acceptor. After s t o p p i n g t h e r e a c t i o n b y h e a t , t h e c o n t e n t of t h e t u b e s w a s desalted, s p o t t e d on W h a t m a n No. i p a p e r a n d c h r o m a t o g r a p h e d in b u t a n o l - p y r i d i n e - w a t e r (6:4:3, wtv) for 20 h; t h e p a p e r w a s dried a n d r e e h r o m a t o g r a p h e d in t h e s a m e s o l v e n t for a n o t h e r 20 h; t h i s process w a s r e p e a t e d once more. A s c a n n i n g for r a d i o a c t i v i t y along t h e p a p e r strip w a s carried o u t w i t h a N u c l e a r Chicago Model D-47 g a s flow c o u n t e r fitted to a C - I o o A actig r a p h I I w i t h 0. 5 i n c h collimator. G: glucose; L,~, l a m i n a r i b i o s e ; L 3, l a m i n a r i t r i o s e ; L 4, l a m i n a r i t e t r a o s e ; L 6, l a m i n a r i p e n t a o s e ; L~, l a m i n a r i h e x a o s e ; L~, l a m i n a r i h e p t a o s e ; L s, l a m i n a r i o c t a o s e . Fig. 8. R a d i o a c t i v e r e a c t i o n p r o d u c t s f r o m b o t h p h o s p h o r y l a s e s u s i n g l a m i n a r i b i o s e as acceptor. (A) Tile c o n d i t i o n s were s i m i l a r to t h o s e described in Fig. 7 C, b u t replacing 0. 5 / z m o l e of l a m i n a r i biose as acceptor. (B) Conditions as described in Fig. 7 B b u t replacing 0. 5/zmole of l a m i n a r i b i o s e for glucose. (C) C o n d i t i o n s similar to (B) b u t after 45 m i n i n c u b a t i o n . O t h e r c o n d i t i o n s a n d a b b r e v i a t i o n s as described in Fig. 7-
(a) Glucose as acceptor. Fig. 7A shows that laminaribiose phosphorylase formed laminaribiose and laminaritriose after short incubation periods; b y increasing the time to 45 rain, laminaritetraose could also be detected (Fig. 7C). The behavior of fl-I,3-oligoglucan phosphorylase was, however, clearly different. Thus, as shown in Fig. 7 B and D, a series of radioactive peaks ranging from laminaribiose to laminariheptaose were easily detected. (b) Laminaribiose as acceptor. Laminaribiose phosphorylase formed laminaritriose, laminaritetraose and traces of laminaripentaose after 45 rain incubation (Fig. 8A) ; the formation of some labeled laminaribiose was also observed, probably Biochim. Biophys. Acta, 146 (1967) 431-442
438
L.R. MARECHAL
due to the reversibility of the reaction. Shorter incubation periods were not run on account of the scarcity of the material produced as judged by the Pi released. On the other hand, fl-I,3-oligoglucan phosphorylase gave the same qualitative pattern as that observed when glucose was used as acceptor, except for the presence of labeled laminaribiose, after either 15 min or 45 min of incubation (Fig. 8B and C, respectively). TABLE III EFFECT OF MERCAPTOETHANOL, IODOAGETATE AND p-MERCURIBENZOATE ON LAMINARIBIOSE AND f l - I , 3 - O L I G O G L U C A N PHOSPHORYLASE ACTIVITY
A: T h e a c t i v i t y of l a m i n a r i b i o s e p h o s p h o r y l a s e was m e a s u r e d as follows: 2 # m o l e s of i m i d a z o l e buffer, p H 7.2, 0.2/~mole of E D T A , a d d i t i o n s in t h e c o n c e n t r a t i o n s i n d i c a t e d a n d o.oi ml of e n z y m e o r i g i n a t e d from t h e first c o l u m n c h r o m a t o g r a p h y (Fig. I) in a final vol. of 0.03 ml. A f t e r p r e i n c u b a t i o n a t 3 °o d u r i n g 20 rain, 5/~moles of glucose a n d i / ~ m o l e of G l c - I - P were a dde d. The m i x t u r e w a s i n c u b a t e d a t 37 ° for 20 mill a n d t h e P i released w a s d e t e r m i n e d . Controls were r u n o m i t t i n g t h e acceptor. B: I n E x p t . i, t h e a c t i v i t y of f l - i , 3 - o l i g o g l u c a n p h o s p h o r y l a s e w a s m e a s u r e d as a b o v e b u t u s i n g o.oi ml of e n z y m e o r i g i n a t e d from t he second c o l u m n c h r o m a t o g r a p h y (1-week-old p r e p a r a t i o n ; see Fig. 2) a n d r e p l a c i n g 0. 5 / , m o l e of l a m i n a r i b i o s e or ] a m i n a r i triose for glucose. I n E x p t . 2, t h e a c t i v i t y of fl-I,3-ol i gogl uc a n p h o s p h o r y l a s e w a s d e t e r m i n e d as d escribed in E x p t . t b u t w i t h a 2 - m o n t h - o l d e n z y m e a n d w h e n i n d i c a t e d 5/*moles of glucose were used. The i n c u b a t i o n was p e r f o r m e d for i o m i n a t 37 °.
Addition
Concerttration
A (l~moles P, released)
B (/~moles t), released)
Glucose
Laminaribiose
Laminaritriose
o.19 o.48 o. 15 0.04 0.06 o
o.17 o.42 o. 12 0.03 0.03 o
o 0.24
o 0.24
(raM) Glucose
Expt. z None Mercaptoethanol Iodoacetate Iodoacetate p-Mercuribenzoate p-Mercuribenzoate
-2o o.2 2 0.007 0.o 7
o.36 o.36 o.37 0. 31 0.37 0.04
Expt. 2 None Mercaptoethanol
-20
o 0.20
Some experiments were carried out using laminaridextrins as acceptors for both enzymes, but the radioactive peaks found were not clearly separated from one another. However, a much lower degree of labeling could be observed with laminaribiose phosphorylase than with/3-1,3-oligoglucan phosphorylase. In another experiment with the latter enzyme, a better separation among the radioactive peaks could be achieved and they were tentatively identified as laminarihexaose, laminariheptaose, laminarioctaose, laminarinonaose and laminaridecaose. Finally, it must be pointed out that in all cases, controls were carried out similarly to the assays but omitting the acceptor. The scanning for radioactivity showed only a small peak in the zone corresponding to glucose, similar to that observed in Figs. 7 and 8. This was due to the presence of free [14C]glucose in the ~14C]Glc-I-P solution, as was demonstrated by paper chromatography. Biochim. Biophys. Acta, 146 (1967) 431-442
/%I,3-GLUCAN PHOSPHORYLASES FROM E.
gracilis.
II
439
Comments From tile quantitative point of view, the results of/~-I,3-oligoglucan phosphorylase activity using glucose as acceptor were rather puzzling when compared with those obtained with laminaribiose as substrate. It can be seen in Fig. 7 B that, in the case of glucose, even after short periods of incubation, the higher oligosaccharides were more heavily labeled than the lower; this effect became more evident after longer incubations (Fig. 7D). In the case of laminaribiose (Fig. 8B and D), as expected, a higher labeling was found in the lower homologues. Further studies on the kinetic behavior of/5-I,3-oligoglucan phosphorylase will be necessary to give a satisfactory explanation for this difference.
Miscellaneous experiments As already stated, sulfhydryl groups seem to be essential for/5-I,3-oligogiucan phosphorylase activity. This requirement was not easily detected in fresh enzyme preparations and was more evident in aged extracts. On the other hand, laminaribiose phosphorylase, in some experiments, was only slightly activated by mercaptoethanol. The experiments described in Table IV were carried out in order to investigate the response of both enzymes to specific inhibitors of - S H groups. I t can be seen t h a t with glucose as acceptor, laminaribiose phosphorylase was only slightly inhibited b y 2 mM iodoacetate, while a 90% inhibition was observed with o.07 mM p-mercuribenzoate*. In the present assay, a higher dilution of the latter inhibitor was not effective. However in other experiments, a 50% inhibition was detected. TABLE IV COMPARISON
OF
DIFFERENT
ACCEPTORS
FOR ~-I,3-OLIGOGLUCAN A N D LAMINARIBIOSE PHOSPHO-
RYLASES In Expt. A, the enzymatic activity was measured as follows: 2/,moles of imidazole buffer, p H 7.2, o.2/*mole of EI)TA, i/,mole of mereaptoethanol (when added) and o.oi ml of $i, 3o l i g o g l u c a n p h o s p h o r y l a s e o r i g i n a t e d f r o m t h e second c h r o m a t o g r a p h y of P e a k I I (see Fig. 2) (8 w e e k s old) were p r e i n c u b a t e d a t 3 ° ° for 3o m i n ; t h e n 0.5/~mole of t h e a e c e p t o r s l i s t e d below a n d I / ~ m o l e of G l c - I - P were a d d e d a n d i n c u b a t e d a t 37 ° for 20 a n d 4 ° min. T h e PI re l e a s e d w a s d e t e r m i n e d as p r e v i o u s l y i n d i c a t e d . I n E x p t . B, t h e c o n d i t i o n s were s i m i l a r to E x p t . A, e x c e p t t h a t o . o i m l of l a m i n a r i b i o s e p h o s p h o r y l a s e o r i g i n a t e d from P e a k I (Fig. I) w a s us e d i n s t e a d of ~ - I , 3 - o l i g o g l u c a n p h o s p h o r y l a s e . Controls were r u n o m i t t i n g t h e acceptors.
Acceptor
fncubation time (min)
M (#moles Pl B (t~moles P~ released) released) --SH +S H --SH +S H
Laminaribiose Glucose a-Phenylglucoside a-Methylglucoside and laminarin
20 4° 20 4° 20 4° 2o 4°
o o o o o o o o
0.40 0.59 o.ii o.28 o.03 o.08 o o
0.03 0.05
0.24 0.45 o o o o
0.06 o.io 0.29 0.47 o o o o
" S e v e r a l e n z y m e b a t c h e s g a v e s i m i l a r r e s u l t s w i t h t h i s c o n c e n t r a t i o n of p - m e r c u r i b e n z o a t e . H o w e v e r i t w a s a b o u t 3o-fold lower t h a n t h a t p r e v i o u s l y r e p o r t e d " t o o b t a i n s i m i l a r i n h i b i t i o n . T h e s e differences could n o t be e x p l a i n e d .
Biochim. Biophys. Acta, 146 (1967) 431-442
440
L. R. MARECHAI
Table IV also shows the results obtained with/~-i,3-oligoglucan phosphorylase. A similar degree of inhibition was obtained with either laminaribiose or laminaritriose as acceptor using iodoacetate or p-mercuribenzoate as inhibitors. These results give additional evidences that the same active center is involved with the two oligosaccharides. For comparison, the effect of a preincubation with mercaptoethanol on the activity of both enzymes is also given. Expt. 2 demonstrates that aged preparations of fl-I,3-oligoglucan phosphorylase were not active in absence of - S H donors using glucose, laminaribiose or laminaritriose as acceptors. Therefore, the results with glucose would support the suggestion that the enzyme preparation used in the experiment described in Fig. 3B was contaminated with some laminaribiose phosphorylase. It might be appropriate to mention at this point that the activity of laminaribiose phosphorylase was not affected when assayed as described under Enzyme assay, Procedure A but substituting 20 mM hydroquinone for mercaptoethanol. On the contrary, the activity of fl-I,3-oligoglucan phosphorylase was completely inhibited under the same conditions. As already mentioned 1, the latter effect was not observed when the enzyme activity was measured in presence of - S H donors. MANNERS AND TAYLOR4 reported that a-methyl- and a-phenylglucosides were poor acceptors for laminaribiose phosphorylase from Astasia ocellata, while laminarin was a good one. However, GOLDEMBERG, MARECHAL AND DE SOUZA2 could not detect any activity with the mentioned substrates and the enzyme from E. gracilis. As previously reported 1, some activity was found with a-phenylglucoside and fl-i,3-oligoglucan phosphorylase. The assay was repeated but using both enzymes separated by DEAE-cellulose chromatography. Table IV shows that of the three mentioned subs~rates assayed only a-phenylglucoside was effective as acceptor and that fl-I,3-oligoglucan phosphorylase was responsible for this activity. Although this result might explain the difference obtained with those given by MANNERS AND TAYLOR4 for a-phenylglucoside, that corresponding to laminarin remains to be solved. In this sense, the presence of another phosphorylase (unpublished results) in crude extracts of E. gracilis which catalyzes the phosphorolysis of laminarin and KOHtreated paramylon, must not be discarded. DISCUSSION So far two/~-I,3-oligoglucan phosphorylases have been found in cell-free extracts from E. gracilis : laminaribiose 2 and/~-I,3-oligoglucan phosphorylases 1. Both of them catalyze the same reaction but can be differentiated by several properties. One of the main characteristics is that /5-I,3-oligoglucan phosphorylase shows a specific requirement for - S H donors as was demonstrated in the previous 1 and present papers. This is more marked with aged enzymatic preparations (see Tables I I I and IV). The kinetic behavior of the enzymes toward glucose or/%I,3-oligoglucans is also different. As shown in Table I, laminaribiose phosphorylase forms Glc-I-P from laminaribiose at a rate approx. 4-fold greater than with higher homologues, while the opposite was observed with the new enzyme. Similar behavior was also detected when laminaribiose was used as acceptor for both enzymes. Fig. 3A shows that the reaction rate of/~-I,3-oligoglucan phospho-
Biochim. Biophys. Acta, 146 (1967) 431-442
~-I,3-GLUCAN PHOSPHORYLASES FROM E. gracilis, ii
44"-
rylase was about 8-fold higher than laminaribiose phosphorylase at optimal concentrations of mercaptoethanol (see also Fig. 4). On the other hand, while laminaribiose phosphorylase retained 9O-lOO% of its activity after 2-3 months l,/~-I,3-oligoglucan phosphorylase generally lost most of its catalytic effect in the same period (however, some exceptions were found, see Table IV). As mentioned previously, one problem that remained to be solved was that related to the phosphorolytic action of laminaribiose phosphorylase on higher oligosaccharides and that corresponding to fl-I,3-oligoglucan phosphorylase on laminaribiose. From the kinetic approach represented in Figs. 5 and 6, it can be concluded that the activities mentioned were due to properties of each enzyme and not to mutual contaminations (see also Table I I I , Expt. 2). It seems interesting to point out that both phosphorylases, and especially /~-I,3-oligoglucan phosphorylase, could form higher oligosaccharides starting with a molecule as small as glucose. As far as it is known, potato phosphorylase uses maltotriose or higher homologues as acceptors, while maltose is ineffective 5. In the case of muscle phosphorylase, maltose appears to be the smallest glucosyl acceptor; however a long incubation time was necessary to detect this result 6. On the other hand, other known disaccharide phosphorylases, sucrose and maltose and cellobiose phosphorylases, can act with fructose and glucose, respectively, as acceptors, but they only form the corresponding disaccharide. All these facts would differentiate the Euglena phosphorylase from that of other organisms. In this sense, it seems interesting that MANNERS AND TAYLOR~ reported the presence of enzyme(s) that catalyze(s) similar reactions in extracts of Ochromonas and Astasia ocellata so that this type of enzyme seems ubiquitous in phytoflagellates. Paramylon is considered to be the reserve carbohydrate of E. gracilis and other Euglenoids. BLUM AND BUETOW7 demonstrated in E. gracilis var. bacillaris that most of the polysaccharide disappears after 13 days starvation, thus indicating the ability of the cells to utilize it as reserve carbon supply. MERRIK AND DOUDOROFFs have found that a very complex mechanism is required to degrade the native poly-~-hydroxybutyric acid granules, the major reserve material of m a n y types of bacteria. They also found that several physical or chemical agents make the grains resistant to digestion. These results could serve as a possible explanation for the fact that several attempts to obtain the in vitro breakdown of native paramylon grains with crude extracts of E. gracilis strain z were unsuccessful. As mentioned before, only a minute amount of Glc-I-P could be detected when these extracts were incubated with laminarin or KOH-treated paramylon. At present, further studies seem to be necessary to elucidate the mechanism that governs the intracellular production of Glc-I-P from paramylon. However it is tempting to ascribe a physiological role to/5-I,3-oligoglucan and laminaribiose phosphorylase. One can assume that the native paramylon granules yield oligosaccharides with a length of 8 - I o glucosyl residues by some unknown mechanism, perhaps b y the action of endoglucanases of the a-amylase type. This molecule is then degraded by /%i,3-oligoglucan phosphorylase giving Glc-I-P b y phosphorolysis as well as an oligosaccharide with one less glucosyl residue. In this way we would get laminaribiose which would be further degraded b y laminaribiose phosphorylase. This hypothetic mechanism is summarized in the following scheme: Biochim. Biophys. Acta, 146 (1967) 431-442
442
L. R. MARECHAI_ P a r a m y l o n - - - - -~- (fl-i,3-Glc) n ? (/3-I,3-Glc)n + Pi ~ ~ a - G l c - i - P + (fl-i,3-Glc)n_ a fl-l,3-oligoglucan phosphorylase (/~-I,3-Glc)n-a + Pi ~-::: ~ a - G l c - I - P + laminaribiose fl- 1,3-oligoglucan p h o s p h o r y l a s e ]aminaribiose + Pi ~ ~ Glc + a - G l c - l - P laminaribiose p h o s p h o r y l a s e
There is some overlapping in the specificity of laminaribiose and fi-I,3-oligoglucan phosphorylases towards the oligosaccharides. However, one might assume that the activity of fl-I,3-oligoglucan phosphorylase on laminaribiose in vivo is insignificant as might be the case of laminaribiose phosphorylase towards the higher homologues, according to their respective in vitro reactions.
ACKNOWLEDGEMENTS
The author wishes to express his gratitude to Dr. LuIs F. LELOIR for his continued guidance and support and to his colleagues at the Instituto de Investigaciones Bioqufmicas for many helpful discussions and criticisms. This work was taken from a thesis to be submitted by the author to the University of Buenos Aires in partial fulfillment of the requirements for the Degree of Doctor of the University of Buenos Aires and was supported in part by a research grant (No. GM 03442) from the National Institutes of Health, U.S. Public Health Service, by the Rockefeller Foundation, and by the Consejo Nacional de Investigaciones Cientfficas y T6cnicas (Argentina). The author is a career investigator of the latter institution. REFERENCES L. R. MARECHAL,Biochim. Biophys. dcla, 146 (i967) 417 . S. H. GOLDEMBERO, L. R. MARECHAL AND B. C. DE SOUZA, J. Biol. Chem., 241 (1966) 45. M. D i x o n AND E. C. WEBB, Enzymes, Longmans, Green and Co., London, 2rid ed., 1964, p. 85. D. J. MANNERS AND ~). C. TAYLOR, Biochem. J., 94 (1965) I7P. D. H. ~BROWN AND C. F. CORI, in P. D. ]~OYER, H. LARDY AND K. MYRB.~.CK, The Enzymes, Vol. 5, Academic Press, N e w York, 2nd e d , 1961, p. 207, 6 ]3. 1LLINGWORTIt, D. H. BROWN AND C. F. CORI, in W. J. WHELAN AND M. P. CAMERON, Ciba Foundation Symposium on Control of Glycogen Metabolism, Churchill, London, 1964, p. lO 7. 7 J. J- BLUM AND D. E. BUETOW, Exptl. Cell Res., 29 (1963) 407 , 8 J. M. MERRICK AND M. DOUDOROFF, J. Bacteriol., 88 (1964) 60. I z 3 4 5
Biochim. Biophys. Acta, 146 (I967) 431-442
431
BBA 65655 fl-I,3-OLIGOGLUCAN : O R T H O P H O S P H A T E GLUCOSYLTRANSFERASES FROM EUGLENA GRACILIS II. COMPARATIVE S T U D I E S B E T W E E N L A M I N A R I B I O S E - AND fl-I,3-OLIGOGLUCAN P H O S P H O R Y L A S E
LUIS R. MARECHAL Instituto de Investigaciones Bioqulmicas "Fundaci6n Campomar", Obligado 2490, Buenos Aires (28) (A rgentina) (Received May i2th, 1967)
SUMMARY
I. Further studies have been carried out on the specificity of laminaribiose- and fl-I,3-oligoglucan phosphorylase. Both enzymes, present in extracts of Euglena gracilis, could be separated either by calcium phosphate gel or by DEAE-cellulose column chromatography. 2. From the data presented it can be concluded that the two enzymes catalyze the same reaction but with different quantitative specificity. The fl-I,3-oligoglucan phosphorylase was found to phosphorolyze laminaritriose and higher homologues at a greater rate than laminaribiose while the opposite behavior was observed with laminaribiose phosphorylase. 3- Another, and probably more striking, difference between the two enzymes was the fact that aged preparations of fl-i,3-oligoglucan phosphorylase showed an absolute requirement of sulihydryl donors while laminaribiose phosphorylase was only slightly activated. 4. The possibility that the qualitative similarity might be due to a crosscontamination of the enzymatic fractions was discarded by competition experiments between substrates and b y the elution pattern of the DEAE-cellulose chromatography.
INTRODUCTION
Two separable enzyme activities on fl-I,3-oligoglucans are present in extracts of Euglena gracilis strain z: laminaribiose phosphorylase (fl-I,3-oligoglucan:orthophosphate glucosyltransferase I) and fl-I,3-oligoglucan phosphorylase (/5-I,3-oligoglucan:orthophosphate glucosyltransferase II). In the preceding communication 1, some properties of the latter enzyme were described, as well as its separation from laminaribiose phosphorylase. Biochim. Biophys. Acta, 146 (1967) 431-4¢2
432
L . R . MARECHAL
Since both phosphorylases catalyze the same reaction, (fl-I,3-Glc)n + Pi ~ (fl-I,3-Glc)n-i + a-D-Glc-I-P
a comparative study between both enzymes has now been carried out in order to gain further insight into their similarities and differences. In addition, a method for the separation of the two enzymes by DEAE-cellulose column chromatography is reported. EXPERIMENTAL PROCEDURE
Most of the materials and methods used were the same as previously described 1 except for those specifically mentioned.
Enzyme assay Unless otherwise specified they were as follows: Laminaribiose phosphorylase. Both Procedures* A and B, detailed in the previous work, have been used except that 5 #moles of glucose were used instead of laminaribiose in Procedure A, and 0.5/,mole of the latter sugar was substituted for laminaritriose in Procedure B. Controls were carried out similarly but omitting the sugars. One unit of enzyme is defined as that amount catalyzing the formation of I/~mole of Pi per min under the conditions described in Procedure A**. fl-z,3-oligoglucan phosphorylase. Either Procedure A or B, described previously i, was used to measure its activity. RESULTS
Enzyme preparation from calcium phosphate gel Cells of Euglena gracilis strain z were grown, harvested and processed as described in the preceding paper i. The first fraction eluted from the calcium phosphate gel with 5 mM sodium pyrophosphate (Fraction I, containing o.oi to 0.04 units) was used as laminaribiose phosphorylase and the first fraction eluted with IO mM sodium pyrophosphate (Fraction IV, containing o.oi to 0.04 units) was employed as fl-I,3oligoglucan phosphorylase.
Enzyme preparation from DEAE-cellulose column chromatography Cells of the same organism grown in darkness were harvested and processed as described previously 2 up to the 33-66% ammonium sulfate step. The latter fraction (13 ml, 50 mg protein per ml) was dialyzed overnight against io mM TrisHCI-I mM EDTA, pH 7.2, and then applied to a column of DEAE-cellulose (16 cm ~ × 32 cm) equilibrated with the same buffer. All operations were carried out at o-4 °. After the protein was passed through the column, the latter was washed with IOO ml of the same buffer, and then with ioo ml of o.i M NaC1 in IO mM Tris-HCl-i mM * The p r e i n c u b a t i o n of laminaribiose p h o s p h o r y l a s e w i t h m e r c a p t o e t h a n o l was n o t necess a r y for activity. However, it was carried out as usual in order to standardize the assay of b o t h phosphorylases. ** This definition of laminaribiose p h o s p h o r y l a s e u n i t is different from t h a t given previously 2.
Biochim. Biophys. Acta, 146 (1967) 431-442
/~-I,3-GLUCAN PHOSPHORYLASES FROM E. gracilis. II
f
peak Z
I
0:10
I .s.,
'
01
w..
433
'
0,f5 . -
' pea/E
020 0.25 NaCI(M)
0.30
'i
0.35
.
o.¢o
.
o~5
.
.
•. . . . .
~20
025
Nac~ (M)
e30
Fig. i. Chromatography on DEAE-cellulose. 13 ml of the "33-66% ammonium sulfate step" fraction2 was applied to a column (16 cmt × 32 cm) equilibrated with o.oi M Tris-HCl-o.ooI M EDTA, pH 7.2. A linear gradient of NaCI between o.I and 0.35 M in the equilibrium buffer was applied to the column (50o ml of each concentration) with a flow rate of 1-1. 5 ml/min. Fractions of approx. 5 ml were collected. The enzyme activity was measured as described in Enzyme assay, Procedure A. Acceptor: (V--IF) glucose; (Ill--B) laminaribiose. Fig. 2. DEAE-cellulose column chromatography of Peak II. 12 ml of the protein extract from Peak II (see text) were passed through a o.75 cm~ × 32 cm column of DEAE-cellulose equilibrated with o.oi M Tris-HCl-o.ooi M EDTA, pH 7.2. A linear gradient of NaC1 between o.i and 0.35 M in the equilibrium buffer (3° ml of each concentration) with a flow rate of 0.3 ml/min. Fractions of approx. 1.5 ml were collected. Symbols as in Fig. I. E D T A , p H 7.2. The protein was eluted with a linear gradient of NaC1 (from o.I M to 0.35 M c o n c e n t r a t i o n in the equilibrium buffer, 500 ml of each saline concentration). The fractions c o n t a i n i n g the peaks of a c t i v i t y were pooled (Fig. I) a n d 2.5 vol, of n e u t r a l s a t u r a t e d a m m o n i u m sulfate solution were added. The suspension was allowed to s t a n d overnight before centrifugation at i o ooo × g for 20 mill. The precipitate o b t a i n e d was dissolved in a small volume of i o mM T r i s - H C l - i mM E D T A buffer, p H 7.2, a n d dialyzed overnight against the same solution. Fig. I shows t h a t two similar a c t i v i t y peaks were o b t a i n e d using glucose as s u b s t r a t e ; one of t h e m (Peak I, laminaribiose phosphorylase) was eluted between o.18 a n d 0.23 M NaC1 a n d the other (Peak II, /5-I,3-oligoglucan phosphorylase) between 0.25 a n d 0.32 M NaC1. W h e n laminaribiose was used as acceptor, two a c t i v i t y peaks were also obtained, b u t the a c t i v i t y of Peak I was very small compared with Peak II. I t can also be observed t h a t the profile of activities o b t a i n e d with the two acceptors were coincident. I n order to discard the possibility t h a t the presence of two a c t i v i t y peaks m i g h t be due to some artifact in the column chromatography, the protein fraction originated from Peak I I was r e c h r o m a t o g r a p h e d in a 0.75 cm ~ × 32 cm DEAE-cellulose c o l u m n prepared as already m e n t i o n e d . Fig. 2 shows t h a t only one peak was o b t a i n e d which emerged at a p p r o x i m a t e l y the same saline c o n c e n t r a t i o n as previously. I t m u s t be m e n t i o n e d t h a t when a n a l y t i c a l DEAE-cellulose columns (0.75 cm 2 × 32 cm) were used, the laminaribiose phosphorylase was eluted between o.14 a n d o.19 M a n d the fi-I,3-oligoglucan phosphorylase emerged between 0.22 a n d 0.26 M NaC1. After the r e c h r o m a t o g r a p h y of Peak II, the active fractions were pooled, c o n c e n t r a t e d a n d dialyzed as m e n t i o n e d above. This p r e p a r a t i o n a n d t h a t corres p o n d i n g to Peak I were used as fi-I,3-oligoglucan phosphorylase a n d laminaribiose phosphorylase, respectively, to carry out some of the assays m e n t i o n e d below.
Comparative studies using enzymes separated with calcium phosphate gel Effect of mercaptoethanol concentration on the reaction rate of both enzymes. The different behaviors of the phosphorylases towards increasing concentrations of
Biochim. Biophys. Acta, 146 (1967) 431-442
434
L.R. MARECHAI
03]A)
~
' ~
1
/
4
1
L
±
_'
"
-
d." 0 2
v
OI
I
t0
I
t
I
I
I
20
30
40
50
60
Mercaptoethonol conch. (mtq)
Fig. 3. Influence of mercaptoethanol concentration on the activity of fl-I,3-oligoglucan and laminaribiose phosphorylase. (A) Laminaribiose (0. 5/,mole) as acceptor. The standard assay for each enzyme was as described in Procedure A, except that the -SH donor concentration was varied as shown. For the assay of fl-i,3-oligoglucan phosphorylase, Fraction IV was used and for that corresponding to laminaribiose phosphorylase, Fraction I was employed. (B) Glucose (5/*moles) as acceptor. The assays were similar to (A) except that another enzyme batch was used, and that the third fraction eluted with 5 mM sodium pyrophosphate (Fraction III) was employed for the assay of /~-i,3-oligoglucan phosphorylase. Symbols as in Fig. I. Solid line: fl-I,3-oligoglucan phosphorylase; broken line: laminaribiose phosphorylase. m e r c a p t o e t h a n o l using laminaribiose as acceptor is shown in Fig. 3A. I t can be seen t h a t t h e reaction rate for fl-I,3-oligoglucan phosphorylase was increased ab o u t Io-fold at o p t i m a l c o n c e n t r a t i o n o f - S H donor, while t h a t for laminaribiose phosphorylase r e m a i n e d unchanged. Similar effects were o b t a i n e d when glucose was used as acceptor (Fig. 3B) b u t only a 2-fold increase in the reaction rate for fl-I,3-oligoglucan phosphorylase was observed. This decrease in the a c t i v a t i o n m i g h t be due to a certain c o n t a m i n a t i o n of the l a t t e r e n z y m e p r e p a r a t i o n with laminaribiose phosphorylase, thus m a s k i n g the ex act a m o u n t of P1 released in absence of m e r c a p t o e t h a n o l (see below). Phosphorolysis of fl-z,3-oligoglucans. B o t h enzymes catalyze t h e phosphorolysis of several/~-I,3-oligoglucans, b u t the initial reaction rates were different. As shown in Table I, laminaribiose phosphorylase act ed on laminaribiose to form a - G l c - I - P , 3 - 4 times faster t h a n on higher oligosaccharides. Th e inverse was true for fl-I,3-oligoglucan phosphorylase. I t m u s t be p o i n t e d out t h a t l a m i n a r i h e p t a o s e was also phosphorolyzed b y laminaribiose phosphorylase. Thus, in an e x p e r i m e n t similar to t h a t described in TABLE I PHOSPHOROLYSIS OF • - I , 3 - O L I G O G L U C A N S
The test conditions were as described in Enzyme assay, Procedure B. The oligosaccharides indicated below (0. 5 #,mole of each) were added and the amount of Gle-I-P formed was measured with the phosphoglucomutase-Glc-6-P dehydrogenase-NADP system.
Substrate
Laminaribiose phosphorylase (mtzmoles Glc-I-P formed per IO rain)
fl-z,3-oligoglucan phosphorylase (mt~moles Glc-z-P formed per io rain)
Laminaripentaose Laminaxitetraose Laminaritriose Laminaribiose
34 44 46 17o
8o
80 80 2O
Biochim. Biophys. Acta, 146 (1967) 431-442
fl-I,3-GLUCAN PHOSPHORYLASESFROM E. gracilis. II
435
Table I and taking the amount of Glc-I-P formed with laminaribiose as IOO%, approx. 3% of the sugar phosphate came from I mM laminariheptaose. Under the same conditions, ~-I,3-oligoglucan phosphorylase formed 13o% of Glc-I-P.
Phosphorolysis of ~-glucosyl derivatives of hydroquinone and saligenin by laminaribiose phosphorylase. It was previously reported 2 that two or more additional products were formed when laminaribiose phosphorylase was incubated with Glc-I-P and several acceptors, as judged by paper chromatography. However, in the case of arbutin, salicin and ~-phenylglucoside only one additional spot was detected. It was suggested that another enzyme could be responsible for the different behavior of these acceptors. A further possibility could be that the higher homologues were not detected due to the scarcity of the material enzymatically produced. A new approach was then attempted taking advantage of the reversibility of the reaction and the higher sensibility of the method for Glc-I-P determination as compared to that for Pi. As shown in Table II, laminaritriosyl-hydroquinone, laminaritetraosyl-saligenin and laminaritriosyl-saligenin were phosphorolyzed by laminaribiose phosphorylase, but the reaction rate was about 9o% lower than for the phenolic monosaccharides. For comparison, the values obtained with laminaribiose and laminaritetraose are also given. Although the results are not conclusive, the possibility remains that laminaribiose phosphorylase is actually involved in the reaction. TABLE
II
P H O S P H O R O L Y S I S OF V A R I O U S S U B S T R A T E S W I T H L A M I N A R I B I O S E P H O S P H O R Y L A S E
The enzyme was assayed as described under Procedure B, except for the glucosyl donors that replace laminaribiose as detailed below. The Gle-I-P formed during the incubation was determined as described in Table I and the amount corresponding to laminaribiose was taken as lOO% of relative activity.
Glucosyl donor
Concentration (raM)
Relative activity
Laminaribiose Laminaribiosyl-hydroquinone Laminaribiosyl-saligenin Laminaritetraose Laminaritriosyl-hydroquinone Laminaritriosyl-saligenin Laminaritetraosyl-sa!igenin
io 8 8 13 8 8 7
IOO 13o
135 i8 IO
5 6
Glucose and several ~-~,3-oligoglucans as acceptors for both enzymes. Since both enzymes could phosphorolyze the same/5-I,3-oligo sugars, although at different rates, it was considered of interest to assay these compounds as acceptors. Fig. 4 shows that laminaribiose phosphorylase and/5-I,3-oligoglucan phosphorylase can actually also catalyze at different rates similar reactions in the direction of synthesis. Comparative studies using enzymes separated with DEAE-cellulose column chromatography Specificity. fl-I,3-oligoglucan phosphorylase originated from the rechromatography on DEAE-cellulose, was assayed with most of the acceptors previously used 1 and similar values for the liberation of Pi were obtained.
Biothim. Biophys. Acta, 146 (1967) 431-442
436
L.R. •
j
,
,
,
,
MARECHA~
,
Q20
•
0.15
~o.m "o .~ 005 0
i
i
I
2
3
4
; ....... i..............'T 5
6
7
Chain length of occeptors (;n glucose units)
Fig. 4. Glucose and several fl-i,3-oligoglucans as acceptors for laminaribiose and fl-i,3-oligoglucan phosphorylases. The enzymatic assays were as described u n d e r Procedure A for the acceptors indicated. The concentration of the sugars was 5 mM, except for laminariheptaose which was 2. 4 mM. After 15 min incubation, the Pl liberation was determined. Symbols and lines as in Fig. 3.
F r o m t h e results of Figs. i a n d 2, it a p p e a r s t h a t b o t h enzymes can use glucose a n d laminaribiose as aceeptor. H o w e v e r to exclude the possibility of a m u t u a l c o n t a m i n a t i o n , c o m p e t i t i o n e x p e r i m e n t s between acceptors were p e r f o r m e d as a new a p p r o a c h to solve this problem. The results are i l l u s t r a t e d in Figs. 5 a n d 6. The t h e o r e t i c a l curves were o b t a i n e d using the following equations according to D i x o n AND WEBB 3 for the d e t e r m i n a t i o n of t h e t o t a l v e l o c i t y (vt) when a single enzyme acts on two s u b s t r a t e s a a n d b Va-
a
b + Vb - K~
Ka
I q'- a/Ka 4- b/Kt,
t
5
-i
1 4 ~
E 40 3o
.~ 2o ~a
~ m
.o O
.....
IO 20 30 40 50 r
,
,,
~
,
,
~
5 fO 15 5~bstrote eqncenttotton (m/q)
,
t
i~---
20
v-v
r
i
i
l
2
4
6
8
m-m
;o ' ;o ' 'oo'!o 70
90 I
Glucose
(mM)
i
l
l
i
i
i
fO
#2
/4
16
1
20
i~
40
Laminoribiose (raM)
Fig. 5 - L a m i n a r i b i o s e phosphorylase. Competition between laminaribiose and laminaritriose. (A) The incubation m i x t u r e s and enzyme activity were carried out as indicated in E n z y m e assay, Procedure B, b u t containing ill addition laminaritriose ( O - - O ) as indicated. Laminaribiose phosphorylase originated from Peak I (Fig. I) (0.o07 units) was used to carry out these experiments. Under the same conditions, the Kra for laminaribiose and laminaritriose were I mM and 2.1 raM, and the Vmax. 4.6 and 1.2 mktmoles/min, respectively. (B) The conditions were similar to (A) b u t m a i n t a i n i n g the a m o u n t of laminaritriose (0. 5/*mole) c o n s t a n t and v a r y i n g t h a t of laminaribiose as indicated. Symbols as in Fig. I. Solid line represents the theoretical curve (see text). Fig. 6. fl-i,3-oligoglucan phosphorylase. Competition between glucose and laminaribiose. (A} The incubation m i x t u r e s and enzyme were carried o u t as indicated in E n z y m e assay, Procedure A, b u t containing glucose as indicated, fl-I,3-oligoglucan p h o s p h o r y l a s e originated from the rec h r o m a t o g r a p h y of Peak I I (0.04 unit) was used to carry out these experiments. U n d e r the same conditions, the K m for glucose and laminaribiose were 45 and 4 raM, and the Vmax. 41 and 47 m#moles/min, respectively. (B) Tile conditions were similar to (A) b u t w i t h 4 ° mM glucose and v a r y i n g the a m o u n t of laminaribiose as indicated. Solid line represents the theoretical curve. Symbols as in Fig. i. 13iochirn. Biophys. Acta, 146 (1967) 431-442
f l - I , 3 - G L U C A N PHOSPHORYLASES FROM E.
gracilis,
437
ii
b are the concentration of substrates, Va and Vb, their m a x i m u m Ka and K~, their Michaelis constants. It can be seen that the experiobtained with both phosphorylases fall on the calculated curves, thus presence of only one active site in each enzymatic preparation. Reaction products with glucose and laminaribiose as acceptors. It seemed interesting to compare the pattern of the reaction products obtained with both phosphorylases in view of their similar enzymic activities. For this purpose, laminaribioseand fl-I,3-oligoglucan phosphorylases were incubated separately with [14qGlc-r-P and glucose or laminaribiose as acceptor. The result of a radiochromatogram scanning after 15 and 45 rain incubation is depicted in Figs. 7 and 8. where a and velocities and mental points indicating the
200 #00
0
-L-AiLed,-
"~200 "tOO 0 200
400 I
0
t0
20 cm
30
40
fO
20 ¢nl
30
40
Fig. 7- R a d i o a c t i v e r e a c t i o n p r o d u c t s f r o m b o t h p h o s p h o r y l a s e s u s i n g glucose as acceptor. (A) L a m i n a r i b i o s e p h o s p h o r y l a s e o r i g i n a t e d f r o m P e a k I (Fig. I) w a s p r e i n c u b a t e d as described in t e x t a n d a f t e r w a r d s i n c u b a t e d w i t h i / ~ m o l e of glucose a n d 0.6/~mole of [14C]Glc-I-P (36 ooo c o u n t s / m i n ) for 15 rain. (B) fl-i,3-oligoglucan p h o s p h o r y l a s e o r i g i n a t e d f r o m t h e r e c h r o m a t o g r a p h y of P e a k II (Fig. 2) w a s p r e i n c u b a t e d as described in t e x t a n d a f t e r w a r d s i n c u b a t e d as detailed in (A) d u r i n g 15 min. (C) Similar to (A) b u t t h e i n c u b a t i o n t i m e w a s increased to 45 min. (D) Similar to (B) b u t t h e i n c u b a t i o n t i m e w a s increased to 45 rain. T h e controls were carried o u t as a b o v e b u t o m i t t i n g t h e acceptor. After s t o p p i n g t h e r e a c t i o n b y h e a t , t h e c o n t e n t of t h e t u b e s w a s desalted, s p o t t e d on W h a t m a n No. i p a p e r a n d c h r o m a t o g r a p h e d in b u t a n o l - p y r i d i n e - w a t e r (6:4:3, wtv) for 20 h; t h e p a p e r w a s dried a n d r e e h r o m a t o g r a p h e d in t h e s a m e s o l v e n t for a n o t h e r 20 h; t h i s process w a s r e p e a t e d once more. A s c a n n i n g for r a d i o a c t i v i t y along t h e p a p e r strip w a s carried o u t w i t h a N u c l e a r Chicago Model D-47 g a s flow c o u n t e r fitted to a C - I o o A actig r a p h I I w i t h 0. 5 i n c h collimator. G: glucose; L,~, l a m i n a r i b i o s e ; L 3, l a m i n a r i t r i o s e ; L 4, l a m i n a r i t e t r a o s e ; L 6, l a m i n a r i p e n t a o s e ; L~, l a m i n a r i h e x a o s e ; L~, l a m i n a r i h e p t a o s e ; L s, l a m i n a r i o c t a o s e . Fig. 8. R a d i o a c t i v e r e a c t i o n p r o d u c t s f r o m b o t h p h o s p h o r y l a s e s u s i n g l a m i n a r i b i o s e as acceptor. (A) Tile c o n d i t i o n s were s i m i l a r to t h o s e described in Fig. 7 C, b u t replacing 0. 5 / z m o l e of l a m i n a r i biose as acceptor. (B) Conditions as described in Fig. 7 B b u t replacing 0. 5/zmole of l a m i n a r i b i o s e for glucose. (C) C o n d i t i o n s similar to (B) b u t after 45 m i n i n c u b a t i o n . O t h e r c o n d i t i o n s a n d a b b r e v i a t i o n s as described in Fig. 7-
(a) Glucose as acceptor. Fig. 7A shows that laminaribiose phosphorylase formed laminaribiose and laminaritriose after short incubation periods; b y increasing the time to 45 rain, laminaritetraose could also be detected (Fig. 7C). The behavior of fl-I,3-oligoglucan phosphorylase was, however, clearly different. Thus, as shown in Fig. 7 B and D, a series of radioactive peaks ranging from laminaribiose to laminariheptaose were easily detected. (b) Laminaribiose as acceptor. Laminaribiose phosphorylase formed laminaritriose, laminaritetraose and traces of laminaripentaose after 45 rain incubation (Fig. 8A) ; the formation of some labeled laminaribiose was also observed, probably Biochim. Biophys. Acta, 146 (1967) 431-442
438
L.R. MARECHAL
due to the reversibility of the reaction. Shorter incubation periods were not run on account of the scarcity of the material produced as judged by the Pi released. On the other hand, fl-I,3-oligoglucan phosphorylase gave the same qualitative pattern as that observed when glucose was used as acceptor, except for the presence of labeled laminaribiose, after either 15 min or 45 min of incubation (Fig. 8B and C, respectively). TABLE III EFFECT OF MERCAPTOETHANOL, IODOAGETATE AND p-MERCURIBENZOATE ON LAMINARIBIOSE AND f l - I , 3 - O L I G O G L U C A N PHOSPHORYLASE ACTIVITY
A: T h e a c t i v i t y of l a m i n a r i b i o s e p h o s p h o r y l a s e was m e a s u r e d as follows: 2 # m o l e s of i m i d a z o l e buffer, p H 7.2, 0.2/~mole of E D T A , a d d i t i o n s in t h e c o n c e n t r a t i o n s i n d i c a t e d a n d o.oi ml of e n z y m e o r i g i n a t e d from t h e first c o l u m n c h r o m a t o g r a p h y (Fig. I) in a final vol. of 0.03 ml. A f t e r p r e i n c u b a t i o n a t 3 °o d u r i n g 20 rain, 5/~moles of glucose a n d i / ~ m o l e of G l c - I - P were a dde d. The m i x t u r e w a s i n c u b a t e d a t 37 ° for 20 mill a n d t h e P i released w a s d e t e r m i n e d . Controls were r u n o m i t t i n g t h e acceptor. B: I n E x p t . i, t h e a c t i v i t y of f l - i , 3 - o l i g o g l u c a n p h o s p h o r y l a s e w a s m e a s u r e d as a b o v e b u t u s i n g o.oi ml of e n z y m e o r i g i n a t e d from t he second c o l u m n c h r o m a t o g r a p h y (1-week-old p r e p a r a t i o n ; see Fig. 2) a n d r e p l a c i n g 0. 5 / , m o l e of l a m i n a r i b i o s e or ] a m i n a r i triose for glucose. I n E x p t . 2, t h e a c t i v i t y of fl-I,3-ol i gogl uc a n p h o s p h o r y l a s e w a s d e t e r m i n e d as d escribed in E x p t . t b u t w i t h a 2 - m o n t h - o l d e n z y m e a n d w h e n i n d i c a t e d 5/*moles of glucose were used. The i n c u b a t i o n was p e r f o r m e d for i o m i n a t 37 °.
Addition
Concerttration
A (l~moles P, released)
B (/~moles t), released)
Glucose
Laminaribiose
Laminaritriose
o.19 o.48 o. 15 0.04 0.06 o
o.17 o.42 o. 12 0.03 0.03 o
o 0.24
o 0.24
(raM) Glucose
Expt. z None Mercaptoethanol Iodoacetate Iodoacetate p-Mercuribenzoate p-Mercuribenzoate
-2o o.2 2 0.007 0.o 7
o.36 o.36 o.37 0. 31 0.37 0.04
Expt. 2 None Mercaptoethanol
-20
o 0.20
Some experiments were carried out using laminaridextrins as acceptors for both enzymes, but the radioactive peaks found were not clearly separated from one another. However, a much lower degree of labeling could be observed with laminaribiose phosphorylase than with/3-1,3-oligoglucan phosphorylase. In another experiment with the latter enzyme, a better separation among the radioactive peaks could be achieved and they were tentatively identified as laminarihexaose, laminariheptaose, laminarioctaose, laminarinonaose and laminaridecaose. Finally, it must be pointed out that in all cases, controls were carried out similarly to the assays but omitting the acceptor. The scanning for radioactivity showed only a small peak in the zone corresponding to glucose, similar to that observed in Figs. 7 and 8. This was due to the presence of free [14C]glucose in the ~14C]Glc-I-P solution, as was demonstrated by paper chromatography. Biochim. Biophys. Acta, 146 (1967) 431-442
/%I,3-GLUCAN PHOSPHORYLASES FROM E.
gracilis.
II
439
Comments From tile quantitative point of view, the results of/~-I,3-oligoglucan phosphorylase activity using glucose as acceptor were rather puzzling when compared with those obtained with laminaribiose as substrate. It can be seen in Fig. 7 B that, in the case of glucose, even after short periods of incubation, the higher oligosaccharides were more heavily labeled than the lower; this effect became more evident after longer incubations (Fig. 7D). In the case of laminaribiose (Fig. 8B and D), as expected, a higher labeling was found in the lower homologues. Further studies on the kinetic behavior of/5-I,3-oligoglucan phosphorylase will be necessary to give a satisfactory explanation for this difference.
Miscellaneous experiments As already stated, sulfhydryl groups seem to be essential for/5-I,3-oligogiucan phosphorylase activity. This requirement was not easily detected in fresh enzyme preparations and was more evident in aged extracts. On the other hand, laminaribiose phosphorylase, in some experiments, was only slightly activated by mercaptoethanol. The experiments described in Table IV were carried out in order to investigate the response of both enzymes to specific inhibitors of - S H groups. I t can be seen t h a t with glucose as acceptor, laminaribiose phosphorylase was only slightly inhibited b y 2 mM iodoacetate, while a 90% inhibition was observed with o.07 mM p-mercuribenzoate*. In the present assay, a higher dilution of the latter inhibitor was not effective. However in other experiments, a 50% inhibition was detected. TABLE IV COMPARISON
OF
DIFFERENT
ACCEPTORS
FOR ~-I,3-OLIGOGLUCAN A N D LAMINARIBIOSE PHOSPHO-
RYLASES In Expt. A, the enzymatic activity was measured as follows: 2/,moles of imidazole buffer, p H 7.2, o.2/*mole of EI)TA, i/,mole of mereaptoethanol (when added) and o.oi ml of $i, 3o l i g o g l u c a n p h o s p h o r y l a s e o r i g i n a t e d f r o m t h e second c h r o m a t o g r a p h y of P e a k I I (see Fig. 2) (8 w e e k s old) were p r e i n c u b a t e d a t 3 ° ° for 3o m i n ; t h e n 0.5/~mole of t h e a e c e p t o r s l i s t e d below a n d I / ~ m o l e of G l c - I - P were a d d e d a n d i n c u b a t e d a t 37 ° for 20 a n d 4 ° min. T h e PI re l e a s e d w a s d e t e r m i n e d as p r e v i o u s l y i n d i c a t e d . I n E x p t . B, t h e c o n d i t i o n s were s i m i l a r to E x p t . A, e x c e p t t h a t o . o i m l of l a m i n a r i b i o s e p h o s p h o r y l a s e o r i g i n a t e d from P e a k I (Fig. I) w a s us e d i n s t e a d of ~ - I , 3 - o l i g o g l u c a n p h o s p h o r y l a s e . Controls were r u n o m i t t i n g t h e acceptors.
Acceptor
fncubation time (min)
M (#moles Pl B (t~moles P~ released) released) --SH +S H --SH +S H
Laminaribiose Glucose a-Phenylglucoside a-Methylglucoside and laminarin
20 4° 20 4° 20 4° 2o 4°
o o o o o o o o
0.40 0.59 o.ii o.28 o.03 o.08 o o
0.03 0.05
0.24 0.45 o o o o
0.06 o.io 0.29 0.47 o o o o
" S e v e r a l e n z y m e b a t c h e s g a v e s i m i l a r r e s u l t s w i t h t h i s c o n c e n t r a t i o n of p - m e r c u r i b e n z o a t e . H o w e v e r i t w a s a b o u t 3o-fold lower t h a n t h a t p r e v i o u s l y r e p o r t e d " t o o b t a i n s i m i l a r i n h i b i t i o n . T h e s e differences could n o t be e x p l a i n e d .
Biochim. Biophys. Acta, 146 (1967) 431-442
440
L. R. MARECHAI
Table IV also shows the results obtained with/~-i,3-oligoglucan phosphorylase. A similar degree of inhibition was obtained with either laminaribiose or laminaritriose as acceptor using iodoacetate or p-mercuribenzoate as inhibitors. These results give additional evidences that the same active center is involved with the two oligosaccharides. For comparison, the effect of a preincubation with mercaptoethanol on the activity of both enzymes is also given. Expt. 2 demonstrates that aged preparations of fl-I,3-oligoglucan phosphorylase were not active in absence of - S H donors using glucose, laminaribiose or laminaritriose as acceptors. Therefore, the results with glucose would support the suggestion that the enzyme preparation used in the experiment described in Fig. 3B was contaminated with some laminaribiose phosphorylase. It might be appropriate to mention at this point that the activity of laminaribiose phosphorylase was not affected when assayed as described under Enzyme assay, Procedure A but substituting 20 mM hydroquinone for mercaptoethanol. On the contrary, the activity of fl-I,3-oligoglucan phosphorylase was completely inhibited under the same conditions. As already mentioned 1, the latter effect was not observed when the enzyme activity was measured in presence of - S H donors. MANNERS AND TAYLOR4 reported that a-methyl- and a-phenylglucosides were poor acceptors for laminaribiose phosphorylase from Astasia ocellata, while laminarin was a good one. However, GOLDEMBERG, MARECHAL AND DE SOUZA2 could not detect any activity with the mentioned substrates and the enzyme from E. gracilis. As previously reported 1, some activity was found with a-phenylglucoside and fl-i,3-oligoglucan phosphorylase. The assay was repeated but using both enzymes separated by DEAE-cellulose chromatography. Table IV shows that of the three mentioned subs~rates assayed only a-phenylglucoside was effective as acceptor and that fl-I,3-oligoglucan phosphorylase was responsible for this activity. Although this result might explain the difference obtained with those given by MANNERS AND TAYLOR4 for a-phenylglucoside, that corresponding to laminarin remains to be solved. In this sense, the presence of another phosphorylase (unpublished results) in crude extracts of E. gracilis which catalyzes the phosphorolysis of laminarin and KOHtreated paramylon, must not be discarded. DISCUSSION So far two/~-I,3-oligoglucan phosphorylases have been found in cell-free extracts from E. gracilis : laminaribiose 2 and/~-I,3-oligoglucan phosphorylases 1. Both of them catalyze the same reaction but can be differentiated by several properties. One of the main characteristics is that /5-I,3-oligoglucan phosphorylase shows a specific requirement for - S H donors as was demonstrated in the previous 1 and present papers. This is more marked with aged enzymatic preparations (see Tables I I I and IV). The kinetic behavior of the enzymes toward glucose or/%I,3-oligoglucans is also different. As shown in Table I, laminaribiose phosphorylase forms Glc-I-P from laminaribiose at a rate approx. 4-fold greater than with higher homologues, while the opposite was observed with the new enzyme. Similar behavior was also detected when laminaribiose was used as acceptor for both enzymes. Fig. 3A shows that the reaction rate of/~-I,3-oligoglucan phospho-
Biochim. Biophys. Acta, 146 (1967) 431-442
~-I,3-GLUCAN PHOSPHORYLASES FROM E. gracilis, ii
44"-
rylase was about 8-fold higher than laminaribiose phosphorylase at optimal concentrations of mercaptoethanol (see also Fig. 4). On the other hand, while laminaribiose phosphorylase retained 9O-lOO% of its activity after 2-3 months l,/~-I,3-oligoglucan phosphorylase generally lost most of its catalytic effect in the same period (however, some exceptions were found, see Table IV). As mentioned previously, one problem that remained to be solved was that related to the phosphorolytic action of laminaribiose phosphorylase on higher oligosaccharides and that corresponding to fl-I,3-oligoglucan phosphorylase on laminaribiose. From the kinetic approach represented in Figs. 5 and 6, it can be concluded that the activities mentioned were due to properties of each enzyme and not to mutual contaminations (see also Table I I I , Expt. 2). It seems interesting to point out that both phosphorylases, and especially /~-I,3-oligoglucan phosphorylase, could form higher oligosaccharides starting with a molecule as small as glucose. As far as it is known, potato phosphorylase uses maltotriose or higher homologues as acceptors, while maltose is ineffective 5. In the case of muscle phosphorylase, maltose appears to be the smallest glucosyl acceptor; however a long incubation time was necessary to detect this result 6. On the other hand, other known disaccharide phosphorylases, sucrose and maltose and cellobiose phosphorylases, can act with fructose and glucose, respectively, as acceptors, but they only form the corresponding disaccharide. All these facts would differentiate the Euglena phosphorylase from that of other organisms. In this sense, it seems interesting that MANNERS AND TAYLOR~ reported the presence of enzyme(s) that catalyze(s) similar reactions in extracts of Ochromonas and Astasia ocellata so that this type of enzyme seems ubiquitous in phytoflagellates. Paramylon is considered to be the reserve carbohydrate of E. gracilis and other Euglenoids. BLUM AND BUETOW7 demonstrated in E. gracilis var. bacillaris that most of the polysaccharide disappears after 13 days starvation, thus indicating the ability of the cells to utilize it as reserve carbon supply. MERRIK AND DOUDOROFFs have found that a very complex mechanism is required to degrade the native poly-~-hydroxybutyric acid granules, the major reserve material of m a n y types of bacteria. They also found that several physical or chemical agents make the grains resistant to digestion. These results could serve as a possible explanation for the fact that several attempts to obtain the in vitro breakdown of native paramylon grains with crude extracts of E. gracilis strain z were unsuccessful. As mentioned before, only a minute amount of Glc-I-P could be detected when these extracts were incubated with laminarin or KOH-treated paramylon. At present, further studies seem to be necessary to elucidate the mechanism that governs the intracellular production of Glc-I-P from paramylon. However it is tempting to ascribe a physiological role to/5-I,3-oligoglucan and laminaribiose phosphorylase. One can assume that the native paramylon granules yield oligosaccharides with a length of 8 - I o glucosyl residues by some unknown mechanism, perhaps b y the action of endoglucanases of the a-amylase type. This molecule is then degraded by /%i,3-oligoglucan phosphorylase giving Glc-I-P b y phosphorolysis as well as an oligosaccharide with one less glucosyl residue. In this way we would get laminaribiose which would be further degraded b y laminaribiose phosphorylase. This hypothetic mechanism is summarized in the following scheme: Biochim. Biophys. Acta, 146 (1967) 431-442
442
L. R. MARECHAI_ P a r a m y l o n - - - - -~- (fl-i,3-Glc) n ? (/3-I,3-Glc)n + Pi ~ ~ a - G l c - i - P + (fl-i,3-Glc)n_ a fl-l,3-oligoglucan phosphorylase (/~-I,3-Glc)n-a + Pi ~-::: ~ a - G l c - I - P + laminaribiose fl- 1,3-oligoglucan p h o s p h o r y l a s e ]aminaribiose + Pi ~ ~ Glc + a - G l c - l - P laminaribiose p h o s p h o r y l a s e
There is some overlapping in the specificity of laminaribiose and fi-I,3-oligoglucan phosphorylases towards the oligosaccharides. However, one might assume that the activity of fl-I,3-oligoglucan phosphorylase on laminaribiose in vivo is insignificant as might be the case of laminaribiose phosphorylase towards the higher homologues, according to their respective in vitro reactions.
ACKNOWLEDGEMENTS
The author wishes to express his gratitude to Dr. LuIs F. LELOIR for his continued guidance and support and to his colleagues at the Instituto de Investigaciones Bioqufmicas for many helpful discussions and criticisms. This work was taken from a thesis to be submitted by the author to the University of Buenos Aires in partial fulfillment of the requirements for the Degree of Doctor of the University of Buenos Aires and was supported in part by a research grant (No. GM 03442) from the National Institutes of Health, U.S. Public Health Service, by the Rockefeller Foundation, and by the Consejo Nacional de Investigaciones Cientfficas y T6cnicas (Argentina). The author is a career investigator of the latter institution. REFERENCES L. R. MARECHAL,Biochim. Biophys. dcla, 146 (i967) 417 . S. H. GOLDEMBERO, L. R. MARECHAL AND B. C. DE SOUZA, J. Biol. Chem., 241 (1966) 45. M. D i x o n AND E. C. WEBB, Enzymes, Longmans, Green and Co., London, 2rid ed., 1964, p. 85. D. J. MANNERS AND ~). C. TAYLOR, Biochem. J., 94 (1965) I7P. D. H. ~BROWN AND C. F. CORI, in P. D. ]~OYER, H. LARDY AND K. MYRB.~.CK, The Enzymes, Vol. 5, Academic Press, N e w York, 2nd e d , 1961, p. 207, 6 ]3. 1LLINGWORTIt, D. H. BROWN AND C. F. CORI, in W. J. WHELAN AND M. P. CAMERON, Ciba Foundation Symposium on Control of Glycogen Metabolism, Churchill, London, 1964, p. lO 7. 7 J. J- BLUM AND D. E. BUETOW, Exptl. Cell Res., 29 (1963) 407 , 8 J. M. MERRICK AND M. DOUDOROFF, J. Bacteriol., 88 (1964) 60. I z 3 4 5
Biochim. Biophys. Acta, 146 (I967) 431-442